A Plasmodium falciparum transcriptional cyclin-dependent kinase-related kinase with a crucial role in parasite proliferation associates with histone deacetylase activity.
ABSTRACT Cyclin-dependent protein kinases (CDKs) are key regulators of the eukaryotic cell cycle and of the eukaryotic transcription machinery. Here we report the characterization of Pfcrk-3 (Plasmodium falciparum CDK-related kinase 3; PlasmoDB identifier PFD0740w), an unusually large CDK-related protein whose kinase domain displays maximal homology to those CDKs which, in other eukaryotes, are involved in the control of transcription. The closest enzyme in Saccharomyces cerevisiae is BUR1 (bypass upstream activating sequence requirement 1), known to control gene expression through interaction with chromatin modification enzymes. Consistent with this, immunofluorescence data show that Pfcrk-3 colocalizes with histones. We show that recombinant Pfcrk-3 associates with histone H1 kinase activity in parasite extracts and that this association is detectable even if the catalytic domain of Pfcrk-3 is rendered inactive by site-directed mutagenesis, indicating that Pfcrk-3 is part of a complex that includes other protein kinases. Immunoprecipitates obtained from extracts of transgenic parasites expressing hemagglutinin (HA)-tagged Pfcrk-3 by using an anti-HA antibody displayed both protein kinase and histone deacetylase activities. Reverse genetics data show that the pfcrk-3 locus can be targeted only if the genetic modification does not cause a loss of function. Taken together, our data strongly suggest that Pfcrk-3 fulfils a crucial role in the intraerythrocytic development of P. falciparum, presumably through chromatin modification-dependent regulation of gene expression.
- SourceAvailable from: Boris Striepen[Show abstract] [Hide abstract]
ABSTRACT: Toxoplasma gondii and Plasmodium falciparum are important human pathogens. These parasites and many of their apicomplexan relatives undergo a complex developmental process in the cells of their hosts, which includes genome replication, cell division and the assembly of new invasive stages. Apicomplexan cell cycle progression is both globally and locally regulated. Global regulation is carried out throughout the cytoplasm by diffusible factors that include cell cycle-specific kinases, cyclins and transcription factors. Local regulation acts on individual nuclei and daughter cells that are developing inside the mother cell. We propose that the centrosome is a master regulator that physically tethers cellular components and that provides spatial and temporal control of apicomplexan cell division.Nature Reviews Microbiology 01/2014; · 22.49 Impact Factor
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ABSTRACT: During the last decade, a vast number of inhibitors, ligands and fluorescent probes have evolved for mammalian protein kinases;however, the suitability of these compounds for studies of evolutionarily divergent eukaryotes has mostly been left beyond the scope of research. Here, we examined whether adenosine analogue-oligoarginine conjugates that hadbeen extensively characterized as efficient inhibitors of thehumanprotein kinases are applicable for targeting Plasmodium protein kinases.We demonstrated that ARCs were not only able to bind to and inhibit a representative member of P.falciparum kinome (cGMP-dependent protein kinase) in biochemical assay, but also affected the general phosphorylation levels in parasites released from the infected red blood cells upon saponin treatment. These findings urge advantaging of already existing biochemical tools, whose initially generic, but intrinsically "tunable" selectivity profiles could be used for dissection of signaling pathways outside the initially defined group of biological targets.Experimental Parasitology 01/2014; · 2.15 Impact Factor
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ABSTRACT: Discovering the mechanisms by which cell signaling controls the cell cycle of the human malaria parasite Plasmodium falciparum is fundamental to designing more effective antimalarials. To better understand the impacts of melatonin structure and function on the cell cycle of P. falciparum, we have synthesized two families of structurally-related melatonin compounds (7-11 and 12-16). All synthesized melatonin analogs were assayed in P. falciparum culture and their antimalarial activities were measured by flow cytometry. We have found that the chemical modification of the carboxamide group attached at C-3 position of the indole ring of melatonin (6) was crucial for the action of the indole-related compounds on the P. falciparum cell cycle. Among the melatonin derivatives, only the compounds 12, 13 and 14 were capable of inhibiting the P. falciparum growth in low micromolar IC50. These results open good perspectives for the development of new drugs with novel mechanisms of action.European journal of medicinal chemistry 03/2014; 78C:375-382. · 3.27 Impact Factor
EUKARYOTIC CELL, June 2010, p. 952–959
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 9, No. 6
A Plasmodium falciparum Transcriptional Cyclin-Dependent Kinase-Related
Kinase with a Crucial Role in Parasite Proliferation Associates
with Histone Deacetylase Activity?†‡
Jean Halbert,1,2Lawrence Ayong,3Leila Equinet,1,2§ Karine Le Roch,1,2¶ Mary Hardy,3Dean Goldring,4
Luc Reininger,1,2Norman Waters,5Debopam Chakrabarti,3and Christian Doerig1,2*
Inserm-EPFL Joint Laboratory, Global Health Institute, Ecole Polytechnique Fe ´de ´rale de Lausanne, CH-1015 Lausanne, Switzerland1;
Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8TA, United Kingdom2; Department of
Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, University of Central Florida, Orlando,
Florida 328263; Department of Biochemistry, School of Biochemistry, Genetics and Microbiology, University of
KwaZulu-Natal, Scottsville, South Africa4; and Australian Army Malaria Institute,
Enoggera, Queensland, Australia 40515
Received 6 January 2010/Accepted 13 March 2010
Cyclin-dependent protein kinases (CDKs) are key regulators of the eukaryotic cell cycle and of the eukaryotic
transcription machinery. Here we report the characterization of Pfcrk-3 (Plasmodium falciparum CDK-related
kinase 3; PlasmoDB identifier PFD0740w), an unusually large CDK-related protein whose kinase domain
displays maximal homology to those CDKs which, in other eukaryotes, are involved in the control of tran-
scription. The closest enzyme in Saccharomyces cerevisiae is BUR1 (bypass upstream activating sequence
requirement 1), known to control gene expression through interaction with chromatin modification enzymes.
Consistent with this, immunofluorescence data show that Pfcrk-3 colocalizes with histones. We show that
recombinant Pfcrk-3 associates with histone H1 kinase activity in parasite extracts and that this association
is detectable even if the catalytic domain of Pfcrk-3 is rendered inactive by site-directed mutagenesis, indicating
that Pfcrk-3 is part of a complex that includes other protein kinases. Immunoprecipitates obtained from
extracts of transgenic parasites expressing hemagglutinin (HA)-tagged Pfcrk-3 by using an anti-HA antibody
displayed both protein kinase and histone deacetylase activities. Reverse genetics data show that the pfcrk-3
locus can be targeted only if the genetic modification does not cause a loss of function. Taken together, our data
strongly suggest that Pfcrk-3 fulfils a crucial role in the intraerythrocytic development of P. falciparum,
presumably through chromatin modification-dependent regulation of gene expression.
Plasmodium falciparum, the protozoan parasite responsible
for the most virulent form of human malaria, causes 1 to 3
millions deaths annually, mostly among children in sub-Sa-
haran Africa. This mortality is expected to rise with the global
emergence and spread of drug-resistant parasites, making the
discovery of alternative control agents an urgent task (43). The
identification of potential targets is now greatly facilitated by
the availability of genomic databases for several species of the
Plasmodium genus (www.plasmoDB.org) (49). Plasmodium
cell cycle regulators represent attractive candidate targets for
intervention, because (i) their activities are most probably es-
sential to parasite survival, and (ii) the overall organization of
the cell cycle in malaria parasites differs considerably from that
in mammalian cells; this is reflected by atypical properties of
the enzymatic machinery controlling cell cycle progression,
suggesting that specific inhibition is achievable (12, 15).
The progression of the eukaryotic cell cycle is tightly con-
trolled by a family of protein kinases, the cyclin-dependent
kinases (CDKs), whose active forms are composed of a cata-
lytic subunit (CDK) and a regulatory subunit (cyclin) (39).
While several mammalian CDKs (CDK1, -2, -3, -4, -6, and -7)
function in cell cycle control, others (CDK8, -9, -10, and -11)
are part of the transcription machinery. CDK7 is a regulator
both of cell cycle progression (through its activity as a CDK-
activating kinase [CAK]) and of transcription (through its ac-
tivity as a component of the general transcription factor
TFIIH) (26). CDK8 and -9 regulate transcription by phosphor-
ylating the C-terminal domain of the large subunit of RNA
polymerase II (2, 18); BUR1, the Saccharomyces cerevisiae
CDK9 homologue previously known as SGV1, has been shown
to regulate transcription through selective control of histone
modifications (7, 17, 30). CDK10 regulates transcription and
cell cycle progression by modulating the activity of the Ets2
transcription factor, a regulator of CDK1 expression (27).
CDK11 interacts with the general precursor mRNA splicing
factors and with RNA polymerase II, thereby playing a role in
transcript production and the regulation of RNA processing
(37). Finally, CDK5 has neuron-specific functions (11).
Among the 85 (or 99, depending on the criteria used for
* Corresponding author. Mailing address: INSERM-EPFL Joint
Laboratory, Global Health Institute, Ecole Polytechnique Fe ´de ´rale de
Lausanne, GHI-SV-EPFL, Station 19, CH-1015 Lausanne, Switzer-
land. Phone: 41 21 693 0983. Fax: 41 21 693 9538. E-mail: christian
† Supplemental material for this article may be found at http://ec
§ Present address: Bureau des valorisations, Universite ´ d’Orle ´ans,
Orle ´ans, France.
¶ Present address: University of California at Riverside, River-
?Published ahead of print on 19 March 2010.
‡ The authors have paid a fee to allow immediate free access to
inclusion) eukaryotic protein kinase (ePK) sequences that
were identified in the P. falciparum kinome (3, 52), 18 clus-
tered within the CMGC group (CDKs, MAPKs [mitogen-ac-
tivated protein kinases], GSK3 [glycogen synthase kinase 3],
and CDK-like), with 6 sequences more closely related to CDKs
than to other CMGC subfamilies. By analogy with their func-
tions in other eukaryotes, and despite the unique characteris-
tics of the Plasmodium cell cycle (13, 31, 41) and transcription
machineries (1, 6, 8–10), it is likely that the Plasmodium CDK-
related kinases play key roles in cell cycle progression and
transcription in the parasite. Among those gene products,
PfPK5 (22, 32, 47), Pfcrk-1 (14), Pfmrk (34, 35, 53), and PfPK6
(5) have been the subjects of biochemical or structural inves-
tigations. However, the only reverse genetics-based informa-
tion published so far regarding the function of CDKs in the
parasite life cycle is that for Pbcrk-1, the orthologue of Pfcrk-1
in Plasmodium berghei, which is essential for erythrocytic
Here we report the functional characterization of pfcrk-3
(PlasmoDB identifier PFD0740w), a gene encoding an unusu-
ally large CDK-related protein (1,339 amino acids) whose ki-
nase domain displays maximal homology to those CDKs which,
in other eukaryotes, are involved in the control of transcrip-
tion. The enzyme associates with a kinase activity present in
parasite extracts, and this association is detectable even if the
catalytic domain of Pfcrk-3 is rendered inactive by site-directed
mutagenesis, suggesting that Pfcrk-3 is part of a complex con-
taining other protein kinases. We demonstrate that Pfcrk-3
interacts with a histone deacetylase (HDAC) in parasite ex-
tracts, and we provide reverse genetics evidence strongly sug-
gesting that the pfcrk-3 gene plays a crucial role in parasite
proliferation during the asexual erythrocytic cycle.
MATERIALS AND METHODS
GST-Pfcrk-3 expression plasmid and site-directed mutagenesis. The Pfcrk-3
catalytic domain was amplified from the 3D7 cDNA clone by using oligonucle-
otides carrying a BamHI (forward primer, CGGGGATCCGATAAAAGAATG
TAAGTTACACA) or a SalI (reverse primer, GGGGTCGACTTATCCTTTTT
GATTACTCTGT) site (underlined). The PCR product was inserted into the
pGEX4T3 plasmid (Amersham Biosciences) at the BamHI and SalI sites. GST-
Pfcrk-3-K445M, a plasmid encoding a mutant glutathione S-transferase (GST)-
Pfcrk-3 fusion protein with an alteration from lysine to methionine at residue
445, was obtained by site-specific mutagenesis using the overlap extension PCR
technique (21). The plasmids were electroporated into Escherichia coli strain
BL21, and the inserts were verified by DNA sequencing prior to protein expres-
Expression and purification of recombinant proteins. GST, GST-Pfcrk-3, and
GST-Pfcrk-3-K445M were induced in Escherichia coli (strain BL21 codon?) with
0.5 mM isopropyl-?-thiogalactopyranoside at 30°C for 4 h. Cells were harvested
and resuspended in ice-cold sonication buffer (phosphate-buffered saline [PBS]
[pH 7.5], 0.1% Triton, 1 mM EDTA, 1 mM dithiothreitol [DTT]) containing
protease inhibitors (1 mM phenylmethylsulfonyl fluoride and Complete mixture
inhibitor tablet from Roche) and 100 ?g/ml lysozyme. After 10 min on ice, the
suspension was sonicated and clarified by centrifugation at 11,000 ? g for 30 min
at 4°C. The resulting supernatant was incubated with glutathione Sepharose resin
(Sigma) for 1 h. The resin was washed four times with sonication buffer and once
with a buffer containing 50 mM Tris-HCl (pH 8.7)–75 mM NaCl. The protein
concentration was determined using the Bio-Rad dye reagent according to the
manufacturer’s recommendations with bovine serum albumin as a standard.
Aliquots of purified proteins were analyzed by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining.
Parasite culture and preparation of parasite extracts. The P. falciparum clone
3D7 was cultured in vitro by standard methods (25). The parasites were grown in
human erythrocytes at 5% hematocrit in complete RPMI 1640 medium in 25-cm2
ventilated flasks. The flasks were kept in a 37°C incubator under 5% CO2. To
remove serum and leukocytes, the human blood was washed three times in RPMI
1640 and the buffy coat removed. The medium was changed daily. Parasitemia
was measured daily by examining Giemsa-stained blood smears and was kept
between 0.5% and 10%.
Fresh or frozen saponin-lysed P. falciparum (3D7) pellets were sonicated in
radioimmunoprecipitation assay (RIPA) buffer (30 mM Tris [pH 8.0], 150 mM
NaCl, 20 mM MgCl2, 1 mM EDTA, 1 mM DTT, 10 ?M ATP, 0.5% Triton
X-100, 1% NP-40, 10 mM ?-glycerophosphate, 10 mM NaF, 0.1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 mM benzami-
dine, and Complete protease inhibitors). Lysates were cleared by centrifugation
at 10,000 ? g for 15 min at 4°C, and the total amount of protein in the super-
natant was measured by the Bio-Rad protein assay.
Pulldown experiments. Glutathione-agarose beads coated with GST, GST-
Pfcrk-3, or GST-Pfcrk-3-K445M were incubated in parasite extracts or in RIPA
buffer alone at 4°C under mild agitation for 1 h (100 ?g of total parasite proteins
for 10 ?g of recombinant proteins on beads). The beads were then washed three
times in RIPA buffer, once in RIPA buffer with 0.1% SDS, and once in a
standard kinase buffer containing 10 mM sodium fluoride, 10 mM ?-glycero-
phosphate, 1 mM phenylmethylsulfonyl fluoride, and Complete mixture protease
inhibitors. Beads were resuspended in a volume of kinase buffer equal to the
volume of beads. A standard kinase assay was then performed in a final volume
of 30 ?l, and the samples were analyzed by SDS-PAGE and autoradiography.
Kinase assays. Kinase assays were performed as described previously (32).
Briefly, reactions (30 ?l) were performed in a standard kinase buffer containing
20 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 2 mM MnCl2, 10 mM ATP, and 5 ?Ci
[?-32P]ATP, using 0.5 ?g of recombinant kinase (or immunoprecipitated mate-
rial [see below]) and 5 ?g of substrate (myelin basic protein [MBP] or histone
H1). After 30 min at 30°C, the reaction was stopped by the addition of Laemmli
buffer, and the reaction product was loaded onto a 12% SDS-polyacrylamide gel.
Following Coomassie blue staining, the gels were dried and exposed for autora-
Antibody production, immunoprecipitation, and immunofluorescence ana-
lyses of wild-type Pfcrk-3. Chicken immunoglobulin (IgY) antibodies against
peptides H2N-CKNRRTLNEDMLSVVD-CONH2 (named VVD) and H2N-P
NERDIKTLRNLPCTN-CONH2 (named PNG), derived from protein se-
quences (residues 539 to 553 and 840 to 855, respectively, encoded by the
PFD0740w gene), were synthesized by Auspep, coupled to rabbit albumin car-
rier, and inoculated into chickens (the PNG peptide was derived from an early
version of PlasmoDB, and the sequence was subsequently changed to PNERD
IKYLRNLPCWN; the two substitutions appear not to have prevented recogni-
tion of Pfcrk-3 by the antibodies [see Results]). The antibodies were isolated and
affinity purified on a peptide-affinity matrix as described previously (19). The
IgYs were used in immunoprecipitation as described previously (38). Briefly, an
anti-Pfcrk-3 antibody bound to protein A Sepharose beads was incubated with an
extract from a parasite at a specific stage (late trophozoite or schizont). Because
of the low affinity of chicken antibodies for protein A Sepharose beads, the
antibody was first incubated with a rabbit anti-chicken antibody before being
coupled to protein A Sepharose beads. After incubation, protein A Sepharose
bead-bound complexes were washed and assayed for kinase activity.
For immunofluorescence analysis, P. falciparum-infected erythrocytes were
fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde as described pre-
viously (51). The cells were probed with chicken anti-Pfcrk-3 (1:400) and mouse
anti-histone (1:500; Chemicon International) and were subsequently incubated
with Alexa Fluor 488-conjugated goat anti-chicken IgY (Molecular Probes) and
Alexa Fluor 555-conjugated goat anti-mouse IgG (Molecular Probes). Confocal
images were acquired using a laser-scanning microscope (LSM 510; Carl Zeiss).
Immunoprecipitation of HA-tagged Pfcrk-3. Parasites expressing hemaggluti-
nin (HA)-tagged Pfcrk-3 or wild-type 3D7 parasites were obtained by saponin
lysis and were then solubilized in M-PER protein extraction reagent (Pierce)
containing 25 U/500 ?l Benzonase and a protease inhibitor cocktail (Roche) for
20 min at 4°C. The lysates were cleared at 9,000 ? g and 4°C for 5 min prior to
immunoprecipitation (IP). For each IP, 500 ?g protein lysate (in a 100-?l total
volume) was incubated with 10 ?l anti-HA agarose slurry (Pierce) overnight at
4°C and was then washed three times, for 5 min each time, with 500 ?l of cold
TBS (25 mM Tris, 0.15 M NaCl [pH 7.2]) buffer.
Histone deacetylase assay. To detect HDAC enzyme activity in the Pfcrk-3
complex, duplicate samples were immunoprecipitated as described above, fol-
lowed by a final wash in HDAC assay buffer (Millipore Corporation). The beads
were further incubated for 16 h at 37°C in 60 ?l HDAC assay buffer containing
100 ?M acetylated fluorogenic peptide and 500 ?M NAD cofactor with or
without HDAC inhibitors (10 mM nicotinamide and/or 2 mM trichostatin A). To
test for the presence of NAD-independent HDAC activities in the Pfcrk-3
immunoprecipitates, duplicate experiments were also performed in the absence
VOL. 9, 2010A P. FALCIPARUM TRANSCRIPTIONAL CDK 953
of the cofactor. The beads were pelleted at 9,000 ? g for 30 s, and 40 ?l of the
supernatant was transferred to each well of a half-volume plate. Twenty micro-
liters of activator solution was added for 15 min, and the resulting fluorescence
was recorded using a plate reader (excitation filter, 360/40 nm; emission filter,
Transfection plasmids. (i) pCAM-BSD-crk-3. A 1,092-bp DNA fragment (nu-
cleotides 1277 to 2368 of the pfcrk-3 open reading frame [ORF]) was amplified
by PCR from P. falciparum genomic DNA, using primers (forward, GGGGGG
ATCCGCATATGGAGATGTTTGGATGGC; reverse, GGGGCGGCCGCTG
GTGGTCTATACCATAATGTAATAACTC) containing BamHI or NotI sites
(underlined). The amplicon was digested with BamHI and NotI and was inserted
at these sites into the pCAM-BSD vector (48).
(ii) pCAM-BSD-crk-3-HA. The pCAM-BSD-HA vector was generated by in-
troducing a sequence encoding a single HA tag and the 3? untranslated region (3?
UTR) of the P. berghei dhfr-ts gene into the multiple cloning site of pCAM-BSD
(see Fig. 5A). The 3? end of the Pfcrk-3 coding region (652 bp, omitting the stop
codon) was amplified by PCR from genomic DNA, using primers with PstI or
BamHI restriction sites, which allowed insertion of the amplified product into the
Parasite transfection and genotype characterization. Ring-stage parasites
were electroporated with 60 ?g of plasmid DNA (pCAM-BSD-crk-3 or pCAM-
BSD-crk-3-HA) as described previously (16). Blasticidin was added to a final
concentration of 2.5 ?g/ml 48 h after transfection. Resistant parasites appeared
4 to 5 weeks posttransfection and were cloned by limiting dilution.
For PCR detection of (i) the integration of the pCAM-BSD-crk-3-HA
plasmid at the 3? flank of the insert, (ii) the integration of the pCAM-BSD-
crk-3 knockout plasmid, and (iii) the episome, the following primers were
used to amplify products from total DNA isolated from parasite lines (see
below): OL-1 (GGTTCATCAAGTTGGACAAGGAGG), OL-2 (CACAAC
TCCACATATCAACCGATGC), OL-3 (TATTCCTAATCATGTAAATCTT
AAA), OL-4 (CAATTAACCCTCACTAAAG), and OL-5 (GAAAGGCTTA
TCTTCGAAGTA). Primers OL-1, OL-2, and OL-5 correspond to pfcrk-3
sequences, while primers OL-3 and OL-4 correspond to pCAM-BSD vector
sequences flanking the insertion site.
For Southern blot analysis, total DNA was obtained as follows. Parasite pellets
obtained by saponin lysis were resuspended in PBS and were treated with 150
?g/ml proteinase K and 2% SDS at 55°C for 2 h. The DNA was precipitated with
ethanol and 0.3 M sodium acetate after phenol-chloroform–isoamyl alcohol
(25:24:1) extraction. The DNA was digested with PstI and SwaI, transferred to a
Hybond N membrane, and hybridized to the pCAM-BSD or pfcrk-3 probe.
Bioinformatic analysis of Pfcrk-3. Phylogenetic analysis of
the P. falciparum kinome (3, 52) identified 18 protein kinases
belonging to the CMGC group, including Pfcrk-3. Among the
different families that constitute the CMGC group, Pfcrk-3
clearly clusters within the CDK family, and more precisely with
the CDK8 to -11 group, which comprises CDKs involved in
transcriptional control (Fig. 1). BLASTP analysis confirmed
the relatedness of Pfcrk-3 to transcriptional CDKs, with the
BUR1 enzyme from yeast giving the highest score (see Discus-
sion). The 11 “invariant” residues that are conserved in most
protein kinases (20, 29) are present in Pfcrk-3, as are the two
signatures that define membership in the protein kinase family
(4) (see Fig. S1 in the supplemental material). The polypeptide
conforms with a high score to the Pfam protein kinase domain
(Pfam entry PF00069; E value, 3.6e?77).
The predicted Pfcrk-3 polypeptide (1,339 amino acids) is
unusually large for a CDK, because the putative catalytic do-
main contains two insertions (of 198 and 20 amino acids), and
there are large N-terminal (378 residues) and C-terminal (335
residues) extensions (see Fig. S1 in the supplemental material).
The extensions and insertions are rich in low-complexity re-
gions and do not display homology to any characterized motif.
The cyclin-binding motif (PSTAIRE in CDK1, PITALRE in
Pfcrk-3. The Thr14 and Tyr15 residues (human CDK2 num-
bering) are the targets of negative regulation by phosphor-
ylation in several mammalian CDKs; only the Tyr15 residue is
present in Pfcrk-3, since Thr14 is replaced by an Ala residue.
Thr160, which is the target of activating phosphorylation by
CDK-activating kinases (CAKs), is conserved in Pfcrk-3.
Expression of pfcrk-3 mRNA and protein in blood stages.
Reverse transcription-PCR (RT-PCR) using primers flanking
the catalytic domain showed that (i) Pfcrk-3 mRNA is present
in asexual and sexual blood stages, (ii) the gene structure (2
exons separated by a 100-bp intron) proposed in PlasmoDB is
correct, and (iii) transcribed mRNA includes the predicted N-
and C-terminal extensions (see Fig. S2 in the supplemental
Microarray data available on PlasmoDB (33) indicate that
pfcrk-3 mRNA is detectable throughout the asexual cycle (as
well as in sporozoites and gametocytes). Northern blot analysis
allowed the detection of a single 4-kb pfcrk-3 mRNA species in
trophozoites, although the signal was very weak (Fig. 2A).
Probing of the same membrane with pfrhoph2 (a gene ex-
FIG. 1. Three-species phylogenetic tree of CDKs. Sequences in
red, blue, and black characters are from P. falciparum, S. cerevisiae, and
Homo sapiens, respectively. Branches with bootstrap values of ?70 are
shown in red, and those with bootstrap values of ?40 are shown in
blue. (Adapted from reference 52.)
FIG. 2. Expression of Pfcrk-3 in asexual blood stages. (A) Northern
blot analysis. Total RNA was extracted from synchronized 3D7 para-
sites (R, rings; T, trophozoites; S, schizonts) and subjected to Northern
blot analysis. (Left) pfcrk-3 probe (catalytic domain); (right) pfrhoph2
probe. (B) Western blot analysis. (Left) The immunopurified IgY
antibody against VVD (a peptide derived from the Pfcrk-3 catalytic
domain) was used to probe extracts from rings, trophozoites, and
schizonts. (Right) An extract from unsynchronized asexual parasites
(U) was probed with immunopurified IgY directed against PNG, the
large insertion within the Pfcrk-3 catalytic domain.
954 HALBERT ET AL.EUKARYOT. CELL
pressed in schizonts) yielded a 7.5-kb mRNA species only in
the schizont stage, as expected (36); equal loading was ascer-
tained by ethidium bromide staining, which yielded rRNA
bands of similar intensities in all lanes (data not shown).
IgY antibodies against two peptides derived from Pfcrk-3
(one in the large insertion and the other in the catalytic domain
[see Fig. S1 in the supplemental material]) recognized recom-
binant Pfcrk-3 in Western blots (data not shown) (see below
for a description of the recombinant protein). Western blotting
performed with extracts from synchronous parasites using the
anti-VVD antibody (directed against the catalytic domain)
showed that although Pfcrk-3 mRNA was detectable predom-
inantly during early stages, the protein was detectable through-
out the asexual cycle (Fig. 2B). Pfcrk-3 appears to be proteo-
lytically processed from a large precursor in rings, whose size
approximates the expected molecular size of the full-length
protein (160 kDa), to a protein of around 120 kDa at later
stages, with lower-intensity bands (including one at 70 kDa)
also detectable (Fig. 2B, left). Determination of the exact pro-
cessing events would require additional studies using antibod-
ies directed against the various parts of the protein. The anti-
body directed against the largest insertion in the catalytic
domain (anti-PNG antibody) (Fig. 2B, right) yielded a similar
profile, suggesting that processing consists of removal of the
In view of the clean Western blot pattern obtained with the
anti-VVD antibody, we next performed immunofluorescence
analysis. Consistent with the Western blot data, the Pfcrk-3
signal was detectable in all intraerythrocytic stages of the par-
asite and largely colocalized with the parasite histone proteins
(Fig. 3). In some ring-stage parasites (e.g., the top row in Fig.
3), Pfcrk-3 appears to localize at the periphery of the nucleus,
a pattern similar to the “horseshoe” pattern observed by Issar
et al. with antibodies against specific histone modifications
Pfcrk-3 is crucial for asexual proliferation. In an attempt to
determine whether or not Pfcrk-3 is essential for erythrocytic
development, P. falciparum 3D7 parasites were transfected
FIG. 3. Pfcrk-3 colocalizes with histones. Images captured by laser
scanning confocal microscopy show substantial colocalization of Pf-
crk-3 (green) with nuclearly localized histones (red). P. falciparum 3D7
parasites were fixed with 4% paraformaldehyde–0.0075% glutaralde-
hyde and were probed with chicken anti-Pfcrk-3 IgY and mouse anti-
histone IgG antibodies. The data in each row represent the fluores-
cence profile in ring-stageparasites,
trophozoites, or schizonts, as indicated. DIC, differential interference
contrast. Bars, 5 ?m in the top row and 2 ?m in all other rows.
FIG. 4. Attempt at disrupting the pfcrk-3 gene. (A) Strategy for
disruption of the pfcrk-3 gene. The transfection plasmid contains a
PCR fragment spanning positions 1277 to 2368 of the 4.2-kb pfcrk-3
coding sequence. This fragment excludes two kinase subdomains es-
sential for activity, labeled GXGXXG (a glycine-rich region required
for correct orientation of ATP) and PE (a proline-glutamate motif in
which the latter residue is required for the structural stability of the
enzyme). Single-crossover homologous recombination results in a
pseudodiploid configuration with two truncated copies, each of which
lacks one of these essential motifs. Oligonucleotides are indicated by
horizontal arrows, restriction sites by vertical lines, and DNA probes
by horizontal bars. BSD, blasticidine deaminase cassette. (B) PCR
analysis of the disrupted locus. Total DNA isolated from cloned, blas-
ticidin-resistant parasites transfected with pCAM-BSD-crk-3 (trans-
fectant) or from wild-type 3D7 parasites (3D7) was subjected to PCR
using the primers indicated (see panel A for their locations). (Left)
Primers OL-3 and -4 (diagnostic for the pCAM-BSD-crk-3 episome or
concatemeric inserts); (center) primers OL-1 and -4 (diagnostic for 5?
integration); (right) primers OL-3 and -2 (diagnostic for 3? integra-
tion). (C) Southern blot analysis. Total DNA was extracted from
cloned blasticidin-resistant parasites transformed with pCAM-BSD-
crk-3 (transfectant) and from wild-type 3D7 parasites (3D7) and was
digested with PstI and SwaI. (Left) After transfer to a Hybond mem-
brane, the digested DNA was probed with the blasticidin resistance
cassette. (Right) The membrane was stripped, and the digested DNA
was probed with a pfcrk-3 amplicon located upstream of the fragment
used as an insert in the pCAM-BSD-crk-3 plasmid. The sizes of comi-
grating markers are given to the left. The band corresponding to the
linearized episome was detected in the transfected parasites (left
panel, left lane). The band corresponding to the intact wild-type locus
was detected in both the transfected and the untransfected parasites
(right panel, both lanes).
VOL. 9, 2010A P. FALCIPARUM TRANSCRIPTIONAL CDK955
with a pCAM-BSD-based construct containing the central por-
tion of the pfcrk-3 gene in order to disrupt the locus (Fig. 4A).
The resulting blasticidin-resistant parasites were then moni-
tored for integration-specific PCR products. No integration-
specific products were observed even after prolonged mainte-
nance of the culture (up to 5 months); the only PCR products
obtained corresponded to the unintegrated episome (Fig. 4B).
Southern blot analysis confirmed the integrity of the wild-type
locus in the transfected cells (Fig. 4C). Failure to disrupt the
pfcrk-3 gene may signify either that the gene is essential for
parasite asexual proliferation or that the vector was unable to
recombine with the locus. To verify that the pfcrk-3 locus was
indeed accessible to recombination, we attempted to modify
the locus without causing loss of function of the gene product.
For this purpose, we transfected wild-type parasites with the
pCAM-BSD-Pfcrk-3-HA plasmid containing the 3? end of the
Pfcrk-3 coding region fused to a hemagglutinin (HA) epitope
followed by the 3? untranslated region (3? UTR) from the P.
berghei dhfr-ts gene (Fig. 5A). Following single-crossover re-
combination, we expect an HA-tagged, functional Pfcrk-3 pro-
tein to be expressed, but we expect no expression from the
wild-type enzyme. PCR analysis of the uncloned blasticidin-
resistant population performed 14 weeks after transfection
readily detected integration of the construct into the pfcrk-3
locus (Fig. 5B, primers OL-5 and -4), in addition to the epi-
some (Fig. 5B, primers OL-3 and -4). Cloned lines were ob-
tained by limiting dilution, and PCR examination of the geno-
types of individual clones at the pfcrk-3 locus demonstrated
that several clones had lost the wild-type locus (data not
shown). The disappearance of the wild-type locus in these
clones and the presence of a modified locus of the expected
size were verified by Southern blot analysis. Figure 5C shows
the data for one such clone, which displays a modified locus
and has either retained the episome or integrated concatemers
of the plasmid.
Association of Pfcrk-3 with protein kinase and histone
deacetylase activities in parasite extracts. In view of (i) the
demonstrated role of BUR1 (the enzyme of the yeast kinome
that is the closest relative to Pfcrk-3) in the regulation of
chromatin modification (including through histone acetyla-
tion) (7) and (ii) the emerging importance of histone deacety-
lases in the regulation of gene expression in Plasmodium (see
reference 50 for a recent contribution to the field), we set out
to investigate whether histone deacetylase activity could be
copurified with Pfcrk-3 from P. falciparum extracts. Unfortu-
nately, the anti-Pfcrk-3 IgYs did not perform satisfactorily in
immunoprecipitation assays (data not shown). We therefore
resorted to the parasite line expressing an HA-tagged version
of Pfcrk-3 (described in the preceding section). We first veri-
fied that an immunoprecipitate obtained with anti-HA anti-
bodies from transgenic, but not from wild-type parasites, con-
tained a protein kinase activity, using mammalian histone H1
(a classical substrate for assaying CDK activity) as a phosphate
receiver. Indeed, kinase activity was much higher in the HA
immunoprecipitate obtained from parasites expressing HA-
tagged Pfcrk-3 than from untransfected 3D7 parasites (Fig.
6A), indicating that Pfcrk-3 is associated, directly or indirectly,
with kinase activity (see below). The autoradiogram displays
many high-molecular-weight bands in addition to the histone
H1 added as a substrate; these may represent copurifying P.
falciparum proteins (including Pfcrk-3 itself) acting as sub-
strates. We then subjected the HA-immunoprecipitated mate-
rial to a histone deacetylase activity assay in the presence or
absence of NAD as a cofactor. As shown in Fig. 6B, no activity
was obtained after immunoprecipitation from extracts of wild-
type parasites (bar 1); only samples from parasites expressing
the HA-tagged protein (bars 2 to 6) exhibited histone deacety-
lase activity. Addition of the cofactor resulted in an increase in
enzyme activity (compare bars 2 and 3), suggesting the pres-
ence of both NAD-dependent and NAD-independent HDAC
activities in the Pfcrk-3-associated complexes. This finding is
consistent with the activity of recombinant PfSir2 as reported
previously (45). Interestingly, the Pfcrk-3-associated HDAC
activities were sensitive to the sirtuin inhibitor nicotinamide
and the class I/II HDAC inhibitor trichostatin A. Together
these data indicate that Pfcrk-3 is part of one or more com-
plexes whose components are capable of protein phosphoryla-
FIG. 5. HA tagging of the pfcrk-3 locus. (A) Strategy for C-termi-
nal tagging of pfcrk-3. The locations of PCR primers used for geno-
typing are indicated by arrows, restriction sites by vertical lines, and
DNA probes by horizontal bars. See the text for details. (B) PCR
analysis of HA-tagged locus. Total DNA isolated from blasticidin-
resistant parasites transfected with pCAM-BSD-crk-3-HA and from
wild-type 3D7 parasites was subjected to PCR using the primers indi-
cated. (Left) Primers OL-3 and -4 (diagnostic for pCAM-BSD-crk-3
episome or concatemeric inserts); (right) primers OL-5 and -4 (diag-
nostic for 3? integration). (C) Southern blot analysis. Total DNA was
extracted from blasticidin-resistant parasites transformed with pCAM-
BSD-crk-3 and from wild-type 3D7 parasites, and 3 ?g was digested
with PstI and SwaI, run on a 0.8% agarose gel, transferred to a Hybond
membrane, and probed with the blasticidin resistance cassette (see
panel A). The membrane was stripped and probed with a pfcrk-3
fragment that is not present in the pCAM-BSD-crk-3-HA plasmid (see
panel A for the location of the probes, which are indicated by hori-
zontal bars underneath the loci). The band corresponding to the lin-
earized episome or concatemeric insert is detected in the transfected
parasites (left panel, lower band). The band corresponding to the
wild-type locus (right panel, right lane) is replaced by a larger band of
the size expected from the recombination of the plasmid into the locus
(both panels, left lanes).
956 HALBERT ET AL.EUKARYOT. CELL
tion and histone deacetylation and that it may play a role in
chromatin modifications by associating with various HDAC
enzymes in P. falciparum.
Kinase activity and pulldown assays with recombinant GST-
Pfcrk-3. In an attempt to demonstrate that Pfcrk-3 itself pos-
sesses kinase activity, a polypeptide containing the catalytic
domain plus the C-terminal extension was expressed in E. coli
as a 100-kDa GST fusion (GST-Pfcrk-3). No kinase activity of
the purified recombinant enzyme was observed under our ex-
perimental conditions, whereas other recombinant P. falcipa-
rum kinases were active (data not shown). We reasoned that
activity of GST-Pfcrk-3 might require interaction with a cyclin-
like activator. Four plasmodial cyclin-like proteins have been
cloned in our laboratory (Pfcyc-1, Pfcyc-2, Pfcyc-3, and Pfcyc-
4), two of which (Pfcyc-1 and Pfcyc-3) activate recombinant
PfPK5 (another CDK-related enzyme) in vitro (38). Incubation
of Pfcrk-3 with these four different recombinant cyclins did not
cause activation of the recombinant enzyme, nor did addition
of RINGO, a potent activator of some CDKs, including PfPK5
(32, 42) (data not shown).
The absence of activity of the recombinant Pfcrk-3 catalytic
domain even in the presence of cyclins may result from the fact
that the fusion protein lacks the large N-terminal extension, or
from the absence of additional activator mechanisms, such as
phosphorylation by other kinases. To address the latter issue,
we repeated the kinase assay following incubation of recombi-
nant Pfcrk-3 in parasite extracts. Pulldown experiments, in
which glutathione-agarose beads loaded with GST-Pfcrk-3
were incubated in extracts from asynchronous parasites,
washed, and subjected to in vitro kinase activity assays, allowed
us to detect histone H1 kinase activity associated with the
recombinant enzyme (Fig. 7A, lane 2). Furthermore, under
these conditions, we also observed a weak signal at approxi-
mately 100 kDa, which is likely to be GST-Pfcrk-3 itself. A
much weaker signal was detected when the pulldown from the
parasite extract was performed with the GST moiety alone
(Fig. 7A, lane 1), and no signal was observed when GST-
Pfcrk-3 was incubated in parasite lysis buffer that did not con-
tain parasite proteins (lane 4). The activity observed in Fig. 7A,
lane 2 (phosphorylation of histone H1 and of GST-Pfcrk-3),
might result either from activation of Pfcrk-3 itself by a com-
ponent of the pulled complex (e.g., through binding of a cyclin-
like regulator or through a phosphorylation event) or from
another protein kinase that had been pulled down by GST-
Pfcrk-3. To distinguish between these possibilities, we repeated
the pulldown experiment using a kinase-dead mutant of GST-
Pfcrk-3 (GST-Pfcrk-3-K445M) in which a conserved lysine res-
idue involved in ATP orientation was replaced by a methionine
residue (Fig. 7B). This yielded a signal of an intensity similar to
that obtained when the pulldown was performed with the wild-
type enzyme (Fig. 7B, lanes 2 and 3), suggesting that another
FIG. 6. Immunoprecipitated HA-Pfcrk-3 associates with protein
kinase and histone deacetylase activities. (A) Association of HA-Pf-
crk-3 with kinase activity. Immunoprecipitates obtained with anti-HA
antibodies from untransfected wild-type 3D7 parasites (lane 1) or from
parasites expressing HA-tagged Pfcrk-3 (lane 2) were subjected to a
kinase activity assay in a standard 30-?l reaction mixture containing 5
?g histone H1 as a substrate. The kinase reactions were then run on a
12% polyacrylamide gel, which was dried and subjected to autoradiog-
raphy. (B) Histone deacetylase assay. Immunoprecipitates obtained
with anti-HA antibodies from untransfected wild-type 3D7 parasites
(bar 1) or from the HA-tagged Pfcrk3-expressing cell line (bars 2 to 6)
were subjected to HDAC assays (Millipore). With the exception of the
experiment for which results are shown in bar 2, the immunoprecipi-
tates were incubated for 16 h at 37°C in a substrate buffer containing
the sirtuin cofactor NAD. HDAC inhibitors (10 ?M nicotinamide
[Nmide] or 2 ?M trichostatin A [TrichoA]) were used separately (bars
4 and 6) or together (bar 5). Reactions were terminated with activator
solution followed by fluorescence measurements (excitation wave-
length, 360 nm; emission wavelength, 460 nm). Error bars represent
standard deviations from duplicate experiments.
FIG. 7. Association of recombinant GST-Pfcrk-3 with histone H1 ki-
nase activity. (A) Pulldown experiments using wild-type GST-Pfcrk-3.
3) bound to glutathione-agarose beads was incubated in parasite extract
(lanes 1 and 2) or in parasite lysis buffer as a negative control (lanes 3 and
4). The beads were washed and resuspended in kinase assay buffer, and in
vitro kinase assays were performed by addition of a reaction mixture
containing radiolabeled ATP and histone H1. (B) Pulldown experiments
using wild-type and kinase-dead GST-Pfcrk-3, followed by kinase assays.
Portions (0.5 ?g) of GST-Pfcrk-3 (lanes 1 and 2), GST-Pfcrk-3-K445M
(lane 3), or GST alone (lane 4) bound to glutathione-agarose beads were
incubated in parasite extracts (lanes 2 to 4) or in parasite lysis buffer as a
negative control (lane 1). The beads were washed, and in vitro kinase
assays were performed by addition of a reaction mixture containing the
radiolabeled ATP and histone H1.
VOL. 9, 2010A P. FALCIPARUM TRANSCRIPTIONAL CDK 957
plasmodial protein kinase that interacts with Pfcrk-3 was re-
sponsible for at least a fraction of the detected kinase activity
identified in Fig. 7A.
Pulldown experiments thus demonstrate that Pfcrk-3 is as-
sociated with kinase activity in parasite extracts and that at
least part of this activity is due to another kinase that interacts
with Pfcrk-3; we favor the hypothesis that Pfcrk-3 itself also
possesses activity in vivo (see Discussion).
Bioinformatics considerations. BLASTP analysis showed
that the Pfcrk-3 kinase domain displays maximal homology to
BUR1 (BLASTP score, 164; E-value, 1e?40), a yeast tran-
scriptional CDK-related kinase previously described as SGV1
and required for recovery from mating pheromone-induced
cell cycle arrest (23). When complexed to its cyclin partner,
BUR2, BUR1/SGV1 phosphorylates the carboxy-terminal end
of RNA polymerase II (40, 54, 55) and other transcription-
related substrates (28) and regulates trimethylation (30). Thus,
the BLASTP results are fully consistent with the phylogenetic
analysis in which Pfcrk-3 clustered with the transcriptional
CDKs, which include SGV1 and human CDK9 (Fig. 1). An-
other feature shared by Pfcrk-3 and BUR1 is a long C-terminal
extension that is usually not found in other CDKs (including
mammalian CDK9); remarkably, this extension has exactly the
same size (335 amino acids) in both enzymes. Even though the
Pfcrk-3 protein appears to be processed during parasite devel-
opment (Fig. 2B), the C-terminal extension is presumably
maintained, since the C-terminally HA-tagged enzyme can be
recovered via the tag (Fig. 6).
Many CDKs are negatively regulated by phosphorylation of
Thr14 and Tyr15 (human CDK2 numbering), of which only
Tyr15 is conserved in Pfcrk-3. Interestingly, the inverse con-
figuration is observed in BUR1 and CDK9, where only Thr14
is conserved; this may indicate different modes of regulation
between Pfrck-3 and transcriptional CDKs in Opisthokhonts
(the phylum including yeast and metazoans). In contrast, the
conservation in Pfcrk-3, yeast BUR1, and human CDK9 of
Thr160, the target of activating phosphorylation by CDK-acti-
vating kinases (CAKs), is consistent with the observation that
BUR1 is activated by CAKs (55) and suggests that a similar
mechanism may regulate Pfcrk-3 activity.
Pfcrk-3 function. Successful in situ 3? HA tagging (Fig. 5)
establishes that the pfcrk-3 locus is recombinogenic, strongly
suggesting that the lack of success in obtaining parasites with a
disrupted locus is due to the fact that the gene is crucial for
asexual proliferation. The possibility remains that Pfcrk-3 in-
activation is not strictly lethal but causes a growth rate defect
that renders parasites unable to compete with parasites that
retain a wild-type locus in the transfected population. In this
context, it is noteworthy that in addition to the inability of
BUR1 mutants to recover from mating pheromone-dependent
cell cycle arrest (23), normal growth under various conditions
is affected in BUR1 and BUR2 mutants (54).
Thus, bioinformatics analyses, subcellular localization, and as-
sociation with histone deacetylase activity all concur to assign
Pfcrk-3 as a chromatin-associated CDK involved in the regulation
of transcription. It would be of great interest, in order to gain
further insight into the precise function of Pfcrk-3, to identify
which of the several putative HDACs (one class I HDAC, two
class II HDACs, and two class III sirtuins ) encoded by the P.
falciparum genome is responsible for the activity we observed to
be associated with HA-Pfcrk-3. Sensitivity to nicotinamide sug-
gests that a class III HDAC, such as Pfsir2, is involved (45), but
caution must be exercised until the enzyme is experimentally
identified. The parasites expressing HA-tagged Pfcrk-3 represent
a tool that can now be used for affinity chromatography/mass
spectrometry-based identification of this and other components
of the protein complex that includes Pfcrk-3. Further information
on the role of Pfcrk-3 in gene expression could be gained by
performing high-resolution colocalization studies to determine
whether the spatial distribution of Pfcrk-3 correlates with that of
specific histone modifications, as hinted by the “horseshoe” ap-
(Fig. 3) (24).
A recombinant protein containing the catalytic domain and the
C-terminal extension, but lacking the N-terminal extension, dis-
played no enzymatic activity. This is expected for a CDK homo-
logue; for example, no activity was observed with the PfPK5 CDK
in the absence of a cyclin (or equivalent) activator (32). However,
in contrast to what we observed with PfPK5, addition of recom-
binant cyclins to the reaction mixture did not result in Pfcrk-1
activation. We showed that a histone H1 kinase activity can be
pulled down from parasite extracts using recombinant Pfcrk-3;
kinase activity was also recovered when a kinase-dead mutant was
is present in a complex that includes Pfcrk-3. Taken together, our
data are consistent with the proposition that Pfcrk-3 functions in
a large complex regulating transcription. In other systems, it is
well established that transcriptional complexes contain several
PKs, including CDKs. If, as we suspect in view of the conservation
of all residues that are important for activity, Pfcrk-3 is indeed an
active CDK, it will of course be crucial for our understanding of
its function to identify substrates of the enzyme. Thepresentstudy
generated the tools necessary to proceed with investigations in these
areas and constitutes a solid basis for further work aimed at under-
standing the control of gene expression in malaria parasites.
This work was supported by INSERM, the European Commission
(FP6 ANTIMAL project and BioMalPar Network of Excellence, and
FP7 MALSIG project and EviMalar Network of Excellence), and a
grant from the Novartis Institute for Tropical Diseases (Singapore).
J.H., L.E., and K.L.R. were supported by studentships from the French
Ministe `re de la De ´fense (De ´le ´gation Ge ´ne ´rale pour l’Armement
[DGA]); D.G.’s work on generating IgYs was funded by the South
African National Research Foundation and Medical Research Coun-
cil; and work in the D.C. laboratory is funded by a grant from the U.S.
National Institutes of Health (AI73795).
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